Misregulation of host cells is a common tactic of bacterial pathogens. In particular, proteins translocated by type III secretion systems (TTSS) encoded by Gram-negative organisms can disrupt a wide range of cellular processes. Many pathogens also encode specialized adhesion systems whose activities, at least superficially, appear unrelated to misregulation. In fact, it is not unusual to find that the activities of the translocated substrates can interfere with the activity of the adhesion protein. Selective pressure clearly has forced maintenance of these supposedly antagonistic proteins, so it is likely that in many cases these proteins collaborate in a fashion that promotes colonization and the establishment of the disease state. It is important to determine how these factors collaborate, because several proteins with very different biochemical activities may act together to alter the regulation of a single downstream target. Such information should allow simplification of drug discovery, as very different proteins that appear to be at war with each other may feed into a single pathway that can be targeted therapeutically. This proposal will focus on the TTSS of Yersinia pseudotuberculosis and its relationship to the outer membrane protein invasin, which promotes bacterial uptake into host cells with concurrent activation of mammalian cell small GTPases. Many of the translocated substrates of the TTSS, called Yops, appear to interfere with invasin function by inactivating these GTPases. Using invasin and YopT, this application will test the hypothesis that, rather than compete with each other, the Yops and invasin collaborate to allow misregulation of host cells. To pursue this goal, the fate of a photoactivated fluorescent derivative of the small GTPase will be followed in response to invasin engagement of host cell receptors. The ability of functional engagement of receptors to form a pool of the GTPase that is readily accessible to YopT will be determined. In addition, a combined photoactivation/photobleaching strategy will be introduced to the microbial pathogenesis field, in order to identify the site in the cell where YopT encounters the small GTPase. In so doing, the model will be tested that bacterial interactions with host cell surface receptors control the site in the cell where YopT ultimately contacts the small GTPase. The long-term goal of these studies is to determine how the entire pool of TTSS substrates cross-regulates the function of other virulence-associated proteins at the site of bacterial adhesion, in a disease-promoting network. The ability to disrupt collaborative bacterial virulence factor interactions that promote the disease process is critical for identifying strategies that maintain immune function in the presence of pathogen attack.